I would like to perform immunofluorescence on CNS tissues, and I was thinking about using IBA-1 to visualize microglia and CD68 to detect peripheral macrophages, however it seems that CD68 it is not so specific. Did anybody have experience in thsi field?
Thank you
Francesca
Ciao Francesca :)! It's very difficult to distinguish between microglia (especially the activated ones) and peripheral macrophages. However, Tmem119 and CD49d should be quite specific for microglia and bone-marrow-derived macrophages (BMDM) respectively. Have a look at Bennet et al., 2016 (for Tmem119) and Bowman et al., 2016 (for CD49d). CD49d alone is not specific for macrophages, so you should always do it in combination with CD11b or Iba1.
Iba1 and CD45 are also good for IHC: on sections you should see microglia as Iba1+ only while macrophages are Iba1+CD45+. However, be careful: microglia cells upregulate CD45 when they get activated! Tmem119 and CD49d should be more specific and restricted even in the activation state according to the original papers.
CD68 is not so specific: it's just less express by resting microglia compared to activated microglia and peripheral macrophages.
Ciao Francesca :)! It's very difficult to distinguish between microglia (especially the activated ones) and peripheral macrophages. However, Tmem119 and CD49d should be quite specific for microglia and bone-marrow-derived macrophages (BMDM) respectively. Have a look at Bennet et al., 2016 (for Tmem119) and Bowman et al., 2016 (for CD49d). CD49d alone is not specific for macrophages, so you should always do it in combination with CD11b or Iba1.
Iba1 and CD45 are also good for IHC: on sections you should see microglia as Iba1+ only while macrophages are Iba1+CD45+. However, be careful: microglia cells upregulate CD45 when they get activated! Tmem119 and CD49d should be more specific and restricted even in the activation state according to the original papers.
CD68 is not so specific: it's just less express by resting microglia compared to activated microglia and peripheral macrophages.
Hi Francesca, besides the excellent advice from Elena, you could try to stain against the purinergic metabotropic receptor P2Y12. It is specific to microglia (and platelets, but that shouldn't be a confound if you perfuse your animal and/or co label with Iba1)
Thank you all for your precious advices and references!!
I know that CD45 is a marker of activation so if microglia is activated it could be easily confused with macrophages, therfore I would avoid using it.
I can try with Tmem119 and CD49d , having a look also to P2Y12 and CD11b.
Thank you
Great advice above! I'll add that Synaptic Systems recently came out with a series of TMEM119 antibodies raised in a variety of species (all of Abcam's are raised in rabbit and most of the good Iba1 antibodies are as well).
I find SYSY to be very reliable in their antibodies.
Check it out: https://www.sysy.com/products/tmem119/index.php
What an informative thread!
May I just add that P2Y12r (alomone labs) labels microglia superbly (processes and cell). However, the labeling is also lost when the microglia become activated (personal observation). This has recently also be demonstrated by others, see O'Koren 2019 https://www.cell.com/immunity/pdf/S1074-7613(19)30073-1.pdf
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